Methods for identification of ligand-blocking antibodies and for determining antibody potency

ABSTRACT

The present disclosure relates to high-throughput systems and methods for the detection of ligand-blocking antibodies and for determining antibody potency.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/965,257 filed Jan. 24, 2020, the disclosures ofwhich are expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. R01AI131722 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

The present disclosure relates to high-throughput systems and methodsfor the detection of ligand-blocking antibodies and for determiningantibody potency.

BACKGROUND

The antibody repertoire—the collection of antibodies present in anindividual—responds efficiently to invading pathogens due to itsexceptional diversity and ability to fine-tune antigen specificity viasomatic hypermutation. This antibody repertoire is a rich source ofpotential therapeutics, but its size makes it difficult to examine morethan a small cross-section of the total repertoire. Historically, avariety of approaches have been developed to characterizeantigen-specific B cells in human infection and vaccination samples. Themethods most frequently used include single-cell sorting withfluorescent antigen baits, screens of immortalized B cells, and B cellculture. However, these methods to couple functional screens withsequences of the variable heavy (V_(H)) and variable light (V_(L))immunoglobulin genes are low throughput; generally, individual B cellscan only be screened against a few antigens simultaneously. What isneeded are high-throughput systems and methods for the detection ofligand-blocking antibodies and for determining antibody potency.

SUMMARY

In some aspects, disclosed herein is a method for simultaneous detectionof an antigen and an antibody that specifically blocks an interactionbetween said antigen and a ligand thereof, comprising:

-   -   labeling a plurality of antigens with unique antigen barcodes;    -   providing a plurality of barcode-labeled antigens to a        population of B-cells to form a mixture;    -   allowing the plurality of barcode-labeled antigens to bind to        the population of B-cells;    -   labeling one or more ligands to one or more antigens in the        plurality of antigens with unique ligand barcodes;    -   introducing the one or more ligands to the mixture of the        plurality of barcode-labeled antigens and the population of        B-cells;    -   washing unbound antigens from the population of B-cells;    -   separating the B-cells into single cell emulsions;    -   introducing into each single cell emulsion a unique cell        barcode-labeled bead;    -   preparing a single cell cDNA library from the single cell        emulsions;    -   performing PCR amplification reactions to produce a plurality of        amplicons, wherein the amplicons comprise: 1) the cell barcode        and the antigen barcode, 2) the cell barcode and an antibody        sequence, and 3) the cell barcode and the ligand barcode, and

wherein each amplicon comprises a unique molecular identifier (UMI);

-   -   sequencing the plurality of amplicons;    -   removing a sequence lacking the cell barcode, the UMI, the        ligand barcode, or the antigen barcode;    -   aligning the antibody sequence to a reference library of        immunoglobulin V, D, J and C sequences;    -   constructing a first UMI count matrix comprising the cell        barcode, the antigen barcode, and the antibody sequence and a        second UMI count matrix comprising the cell barcode, the ligand        barcode, and the antibody sequence;    -   determining a first LIBRA-seq score according to the first UMI        count matrix and a second LIBRA-seq score according to second        UMI count matrix; and    -   determining that the antibody blocks the interaction between the        antigen and the ligand if the first LIBRA-seq score is higher in        comparison to a first reference level and the second LIBRA-seq        score is lower in comparison to a second reference level.

In some embodiments, the barcode-labeled antigens are labeled with afirst barcode comprising a DNA sequence or an RNA sequence. In someembodiments, the cell barcode-labeled beads are labeled with a secondbarcode comprising a DNA sequence or an RNA sequence.

In some embodiments, the antibody sequence comprises an immunoglobulinheavy chain (VDJ) sequence, or an immunoglobulin light chain (VJ)sequence.

In some embodiments, the barcode-labeled antigens comprise an antigenfrom a pathogen or an animal In some embodiments, the antigen is notpurified. In some embodiments, the antigen from a pathogen comprises anantigen from a virus. In some embodiments, the antigen from a viruscomprises an antigen from human immunodeficiency virus (HIV), an antigenfrom influenza virus, or an antigen from respiratory syncytial virus(RSV).

In some embodiments, the method of any preceding aspect furthercomprises determining a level of somatic hypermutation of the antibodyspecifically binding to the antigen.

In some embodiments, the method of any preceding aspect furthercomprises determining a length of a complementarity-determining region(CDR) of the antibody specifically binding to the antigen.

In some embodiments, the method of any preceding aspect furthercomprises determining a motif of a CDR of the antibody specificallybinding to the antigen. In some embodiments, the CDR is selected fromthe group consisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

In some aspects, disclosed herein is a method for simultaneouslyscreening an antigen and an antibody that specifically binds saidantigen, comprising:

-   -   generating a plurality of antigens using an antigen display        technology, wherein each of the plurality of antigens is linked        to a nucleic acid sequence that identifies a particular antigen;    -   providing the plurality of antigens to a population of B-cells;    -   allowing the plurality of antigens to bind to the population of        B-cells;    -   washing unbound antigens from the population of B-cells;    -   separating the B-cells into single cell emulsions;    -   introducing into each single cell emulsion a unique cell        barcode-labeled bead;    -   preparing a single cell cDNA library from the single cell        emulsions;    -   performing PCR amplification reactions to produce a plurality of        amplicons, wherein the amplicons comprise: 1) the cell barcode        and the nucleic acid sequence that identifies the particular        antigen, and 2) the cell barcode and an antibody sequence, and        wherein each amplicon comprises a unique molecular identifier        (UMI);    -   sequencing the plurality of amplicons;    -   removing a sequence lacking the cell barcode, the UMI, or the        nucleic acid sequence that identifies the particular antigen;    -   aligning the antibody sequence to a reference library of        immunoglobulin V, D, J and C sequences;    -   constructing a UMI count matrix comprising the cell barcode, the        nucleic acid sequence that identifies the particular antigen,        and the antibody sequence;    -   determining a LIBRA-seq score;    -   determining the nucleic acid sequence that identifies the        particular antigen; and    -   determining that the antibody specifically binds an antigen if        the LIBRA-seq score of the antibody for the antigen is higher        than a reference level.

In some embodiments, the antigen display technology comprises a ribosomedisplay technology.

In some aspects, disclosed herein is a method for determining a bindingpotency of an antibody to an antigen, comprising:

-   -   labeling a plurality of antigens with unique antigen barcodes;    -   providing a plurality of barcode-labeled antigens to a        population of B-cells;    -   allowing the plurality of barcode-labeled antigens to bind to        the population of B-cells;    -   washing unbound antigens from the population of B-cells;    -   separating the B-cells into single cell emulsions;    -   introducing into each single cell emulsion a unique cell        barcode-labeled bead;    -   preparing a single cell cDNA library from the single cell        emulsions;    -   performing PCR amplification reactions to produce a plurality of        amplicons, wherein the amplicons comprise: 1) the cell barcode        and the antigen barcode, and 2) the cell barcode and an antibody        sequence, and wherein each amplicon comprises a unique molecular        identifier (UMI);    -   sequencing the plurality of amplicons;    -   removing a sequence lacking the cell barcode, the UMI, or the        antigen barcode;    -   aligning the antibody sequence to a reference library of        immunoglobulin V, D, J and C sequences;    -   constructing a UMI count matrix comprising the cell barcode, the        antigen barcode, and the antibody sequence;    -   determining a LIBRA-seq score; and    -   determining that the antibody has a high binding potency to the        antigen if the LIBRA-seq score of the antibody for the antigen        is higher than a reference level.

DESCRIPTION OF DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate aspects described below.

FIG. 1 shows LIBRA-seq for recombinant soluble antigens that are notpurified. Microexpression of antigens occurs in a plate format. A uniqueDNA-barcode is added to each well, and then barcoded antigens are pooledand mixed with cells of interest for LIBRA-seq analysis.

FIG. 2 shows LIBRA-seq for antigens that are not in soluble, recombinantform. The left panel shows that whole virus is tagged with a DNA-barcodeand used for LIBRA-seq analysis. The right panel shows that pseudoviruscan contain an internal barcode and used for LIBRA-seq analysis.

FIG. 3 shows LIBRA-seq in a plate-based single-cell format. Cells ofinterest are isolated and then mixed with an antigen screening librarycomposed of DNA-barcoded, fluorescently labeled antigens.Antigen-positive cells are single cell sorted by fluorescence activatedcell sorting into individual wells of a plate. Cells are lysed and Bcell receptor heavy and light chain variable genes along withantigen-oligo sequences are amplified by PCR using primers containingplate and well specific primers. Amplicons are pooled and sequenced.

FIG. 4 shows LIBRA-seq in microwell format. After staining cells with anantigen library of DNA-barcoded, fluorescently labeled antigens, antigenpositive cells are sorted using fluorescence activated cell sorting.Single cells are deposited into individual, isolated microwellscontaining reagents and primer beads on a microwell array throughgravity and LIBRA-seq is carried out.

FIG. 5 shows LIBRA-seq for antibody discovery with ligand blocking: forBCRs on a B cell.

FIG. 6 shows LIBRA-seq for antibody discovery with ligand blocking: forBCRs on a B cell. The left panel shows traditional LIBRA-seq pipelineidentifies antigen specificity via sequencing of unique antigenbarcodes. Some antibodies function by blocking interactions between aprotein antigen and a ligand. To identify antibodies such as these, abarcoded ligand is used as part of the LIBRA-seq antigen panel. Uponsequencing, B cells that have a high LIBRA-seq score for the givenantigen but a low LIBRA-seq score for the corresponding ligand mayindicate antigen-ligand binding may be decreased when the antigen isbound to the BCRs for the given B cell.

FIG. 7 shows LIBRA-seq for high-throughput antigen screening and de novoantigen discovery. The LIBRA-seq pipeline is conducive to coupling withhigh-throughput antigen screening methods, such as ribosome display.Pre-defined or randomly generated screening libraries displayed onribosomes can be mixed with cell populations of interest and sequenced.In this example, mRNA sequences from each bound ribosome areincorporated with the cellular barcode, along with all other cellulartranscripts, for bioinformatic mapping of B cell receptor sequence todisplayed open reading frames (ORFs).

FIG. 8 shows LIBRA-seq for antibody-antigen potencydetermination—qualitative potency estimates. VRCO1 Ramos B cells andFe53 Ramos B cells were mixed with an antigen screening library composedof BG505 and three epitope mutants of BG505 (N160K, K169E, D358R) andHA. Overall, VRCO1 cells showed lower scores for BG505 D368R compared toBG505 wt, BG505 K169E, and BG505 N160K, in agreement with the decreaseaffinity of VRCO1 for the D368R mutant compared to wildtype. These dataindicate that LIBRA-seq can be used to make qualitative potencyestimates for an antibody and antigen.

FIG. 9 shows LIBRA-seq for antibody-antigen potencydetermination—quantitative potency estimates. To quantitatively assessantibody-antigen potency, several aliquots of the same antigen areindependently labeled with different unique barcodes and then mixed atdifferent fold dilutions with B cells. Upon performing LIBRA-seq, apseudo-potency measurement is obtained for the B cells by fitting acurve to the fold-dependent LIBRA-seq scores for the given antigen.

FIG. 10 is a LIBRA-seq assay schematic showing LIBRA-seq withpre-filtering for antigen-bound B cells. Cells of interest are prepared.For example, donor PBMCs are isolated from blood. Then, PBMCs arestained with a panel of DNA-barcoded, fluorescently labeled antigens.DNA-barcoded antigens are labeled with fluorescently labeledstreptavidin. (Alternatively, fluorescently labeled oligo barcodes canbe used in the antigen-oligo barcoding. In this way, antigens arefluorescently labeled for pre-filtering using fluorescence activatedcell sorting(FACS)). Fluorescently labeled antigens are mixed with cellsof interest. Antigen positive cells are sorted via FACS prior to singlecell sequencing. Single cell suspensions of antigen positive cells areprocessed and sequenced.

FIGS. 11A and 11B show LIBRA-seq with pre-filtering for antigen-bound Bcells. FIG. 11A shows using the LIBRA-seq with pre-filtering for antigenbound B cells. An experiment was performed with a three-antigenscreening library (BG505, CZA97 and HA) on VRCO1 Ramos B cell lines andFe53 Ramos B cell lines. Cells were mixed with DNA-barcoded antigenslabeled with streptavidin-PE and sorted for antigen positivity. Thesesingle cell suspensions were processed and sequenced. LIBRA-seq scoresfor each antigen are shown and each cell is plotted based on thesescores. Shown are density plots from low to high. Cells fell into twopopulations based on their LIBRA-seq scores. FIG. 11B shows VRCO1 cells,and cells displayed high LIBRA-seq scores for both BG505 and CZA97.

FIG. 12 shows the gating scheme for LIBRA-seq with pre-filtering forantigen bound B cells applied to HIV-infection sample from donor N90.PBMCs from donor N90 were mixed with a nine-antigen screening librarycomposed of HIV trimers and influenza trimers, each labeled withstreptavidin PE. Cells were gated on forward scatter and side scatter.Then cells were gated for singlets on side scatter width and height.Cells were further gated for singlets based on forward scatter width andheight. Singlets were gated as Live/CD14−/CD3−/CD19+. From theLive/CD14−/CD3−/CD19+ population, antigen positive cells were sorted andenriched for IgG+. An antigen-PE fluorescence minus one control was alsoincluded.

FIGS. 13A-13B show LIBRA-seq antigen titration for identification ofpotent antibodies. To create affinity-type measurements and identifyhigh potency antibodies using the LIBRA-seq technology, an antigenscreening library containing an antigen titration was applied. Sixdifferent amounts of oligo-labeled SARS-CoV-2 S protein were included ina screening library. Antibodies with high affinity for SARS-CoV-2 Sshowed reactivity for S protein added in lower amounts. FIG. 13A shows aschematic depicting the experimental set up - where a titration ofoligo-labeled S protein was added to the antigen library and donor PBMCswere used as the cellular input. After incubation, cells with highaffinity for the antigen would have many S proteins bound, includingthose added in low concentrations. FIG. 13B shows, after single cellprocessing and sequencing, antigen binding can be assessedbioinformatically and which cells have high LIBRA-seq scores for many orall of the Spike antigens included were determined.

FIG. 14 shows assessment of ligand blocking functionality usingLIBRA-seq through identification of ACE2 blocking antibodies. Forassessment of ligand blocking functionality using LIBRA-seq, an antigenand its ligand are included in the screening library. If an antibodydoes not disrupt the interaction between a protein and its receptor,then the LIBRA-seq scores for the protein and the receptor are high(left). If an antibody does block the interaction, then the score forthe protein is high and the score for the receptor is low (right). Thisallows for identification of antibodies that block receptor binding.This can also indicate neutralization potential of the antibodies. Thisschematic depicts this experimental rationale using SARS-CoV-2 as anexample-where oligo labeled spike and oligo-labeled ACE2 (the spikereceptor) are included in the antigen screening library.

FIGS. 15A-15B show LIBRA-seq antigen titration with ligand blocking foridentification of potent antibodies. In this schematic, an antigentitration along with the inclusion of the receptor are included toidentify potent antibodies with ligand blocking functionality. FIG. 15Ashows schematic depicting the experimental set up—where a titration ofoligo-labeled S protein was added to the antigen library along witholigo-labeled ACE2 receptor, and donor PBMCs were used as the cellularinput. After incubation, cells with high affinity for the antigen wouldhave many S proteins bound, including those added in low concentrations.Antibodies that can block the receptor-protein interaction would nothave ACE2 bound to the spike proteins. Antibodies that do not block theinteraction would have ACE2 bound to the spike proteins. FIG. 15 shows,after single cell processing and sequencing, assessment of antigenbinding bioinformatically and determination regarding which cells havehigh LIBRA-seq scores for many or all of the Spike antigens included.Additionally, which cells do or do not have ACE2 bound can bedetermined. In this example, ACE2 is not bound to spike and thereforehas a low LIBRA-seq score, indicating that the antibody is able to blockligand binding.

FIG. 16 shows extending LIBRA-seq technology for identification ofpotent SARS-CoV-2 antibodies. To assess affinity measurements and ligandblocking functionality, three LIBRA-seq experiments were performed. Toassess affinity measurements, in experiment 1, the antigen libraryconsisted of an antigen titration of SARS-CoV-2 S protein along withcontrol antigens influenza HA NC99 and HIV ZM197. To assess ligandblocking, in experiment 2, the antigen library consisted of SARS-CoV-2 Sprotein along with its receptor, ACE2, and control antigens influenza HANC99 and HIV ZM197. To assess affinity measurements in combination withligand blocking, in experiment 3, the antigen library consisted of anantigen titration of SARS-CoV-2 S protein, ACE2, and control antigensinfluenza HA NC99 and HIV ZM197. Each antigen library was incubated withSARS-CoV-2 convalescent donor PBMCs and LIBRA-seq was performed. Aftersingle cell processing, next generation sequencing, and bioinformaticanalysis, antibody heavy chain and light chain sequence features andantigen LIBRA-seq scores for thousands of cells were assessed. For theantigen titration experiments, antibodies that showed high scores for Sprotein added in lower amounts were identified. For ligand blocking,antibodies that had high scores for S protein and low scores for ACE2were identified—showing ligand blocking functionality of theseantibodies. Antibodies were prioritized for expression and furthertesting based on these features (see FIG. 17 ).

FIGS. 17A-17C show LIBRA-seq enabled prioritization of antibodies withdiverse sequence features and functional profiles using antigentitration and ligand blocking features. As described in FIG. 16 , threeexperiments were performed to assess affinity measurements and ligandblocking in the context of SARS-CoV-2. Antibodies were prioritized forexpression and characterization utilizing the genetic features of theheavy and light chain sequences (including clonal expansion, VH geneusage, VH identity, CDRH3 sequence and sequence length, VL gene usage,VL identity, CDRL3 sequence and sequence length) and the LIBRA-seqscores for the antigens used in each library. For each experiment,select prioritized antibodies are shown, with their genetic features andLIBRA-seq scores. Each row represents an antibody. LIBRA-seq scores foreach antigen in the library are displayed as a heatmap, with LIBRA-seqscore of −2 displayed as tan, a score of 0 displayed as white, and ascore of 2 displayed as purple These antibodies were expressed,purified, and characterized for binding to SARS-CoV-2 S and SARS-CoV-1 S(shown as ELISA area under the curve (AUC)), and neutralization ofSARS-CoV-2. ELISA binding data against the antigens are displayed as aheatmap of the AUC analysis, with AUC of 0 displayed as white, andmaximum AUC as purple. Neutralization is shown as weak, partial orstrong, as green, yellow and red respectively. Non-neutralizingantibodies are listed as white. Additionally, epitope mapping wasperformed by testing binding to a variety of S protein subdomains, anddetermined epitopes are listed. ND stands for not done. HP stands forhexapro and represents the SARS-CoV-2 hexapro S variant that was used inthe screening library. FIG. 17A shows that nine antibodies wereprioritized and tested from experiment 1 (assessment of affinitymeasurements using antigen titration). FIG. 17B shows that tenantibodies were prioritized and tested from experiment 2 (assessment ofligand blocking). FIG. 17C shows that eleven antibodies were prioritizedand tested from experiment 3 (assessment of affinity measurementscombined with ligand blocking). In addition to the select antibodieshighlighted here, there are thousands of other antibodies present in thedatasets. The sequences in FIG. 17A are CARDPASYYDFWSGYVDYYYYGMDVW (SEQID NO: 1), CARDPASYYDLWSGYVDYYYYGMDVW (SEQ ID NO: 2), CARSGGYRLWFGELW(SEQ ID NO: 3), CAREGAVGATSGLDYW (SEQ ID NO: 4), CARGFDYW (SEQ ID NO:5), CARGAGEQRLVGGLFGVSHFYYYMDVW (SEQ ID NO: 6), CAKSATIVLMVSAIYW (SEQ IDNO: 7), CARVRGGEWVGDLGWYYYYGMDVW (SEQ ID NO: 8), CVKGATKIDYW (SEQ ID NO:9), CQQYGNSRLTF (SEQ ID NO: 10), CHHYGSSRLTF (SEQ ID NO: 11),CQQYGGSPATF (SEQ ID NO: 12), CYSRDSSGNPLF (SEQ ID NO: 13), CQQYGSSPWTF(SEQ ID NO: 14), CQQYNSYPWTF (SEQ ID NO: 15), CSSYTSTSTLVF (SEQ ID NO:16), CMQALQTPRTF (SEQ ID NO: 17), CFSYTSGGTRVF (SEQ ID NO: 18). Thesequences in FIG. 17B are CAADPFADYW (SEQ ID NO: 19), CARGLWFGDSETVWFDPW(SEQ ID NO: 20), CVKGKIQLWLGADYW (SEQ ID NO: 21), CARKPLLHSSVNPGAFDIW(SEQ ID NO: 22), CAREKGYSSSSSATYYLDFW (SEQ ID NO: 23), CARRVPGDYYCLDVW(SEQ ID NO: 24), CARGGLWGTFDYW (SEQ ID NO: 25), CARAYGGNYYYGMDVW (SEQ IDNO: 26), CASLGGDSYISGTHYDRSGYDPW (SEQ ID NO: 27), CARVNRVGDGPDFW (SEQ IDNO: 28), CATWDDSLNAWVF (SEQ ID NO: 29), CQQSYSTPPTF (SEQ ID NO: 30),CQQSYNTPWTF (SEQ ID NO: 31), CQQYATSPRTF (SEQ ID NO: 32), CQSYDSSLTALVF(SEQ ID NO: 33), CQQSFSARVPTF (SEQ ID NO: 34), CQQFAYSLYTF (SEQ ID NO:35), CQAWDSSTASFVF (SEQ ID NO: 36), CQRRSNWPPFTF (SEQ ID NO: 37),CMQALQTPWTF (SEQ ID NO: 38). Sequences in FIG. 17C shows CTRGGWPSGDTFDIW(SEQ ID NO: 39), CAREGGWYSVGWVDPW (SEQ ID NO: 40), CARDRRIIGYYFGMDVW(SEQ ID NO 41):, CARLLIEHDAFDIW (SEQ ID NO: 42), CAREEGSGWWKHDYW (SEQ IDNO: 43), CVRDRRIVGYYFGLDVW (SEQ ID NO: 44), CAKDAFYYGSGSHFYYYYYMDVW (SEQID NO: 45), CARDRRGGGWTASFDFW (SEQ ID NO: 46), CARGGWPSGDTFDIW (SEQ IDNO: 47), CAHHTVPTIYDYW (SEQ ID NO: 48), CAKDIGRYDHYNIFGRVGGAFDIW (SEQ IDNO: 49), CQQYGSSRTF (SEQ ID NO: 50), CCPYADTWVF (SEQ ID NO: 51),CMQALHFPYTF (SEQ ID NO: 52), CQQLSGYPYTF (SEQ ID NO: 53), CCSYATTWVF(SEQ ID NO: 54), CQQYGSSPTF (SEQ ID NO: 55), CQQHYSTPGYTF (SEQ ID NO:56), CQQLNSYPEITF (SEQ ID NO: 57), CSSYAGSNPLVF (SEQ ID NO: 58),CQHYDNLPRF (SEQ ID NO: 59),

FIGS. 18A-18C show identification of SARS-CoV-2 antibodies usingLIBRA-seq antigen titration. Utilizing an antigen titration can lead toaffinity-type measurements. By plotting the LIBRA-seq score for the Santigens against the amounts of antigen that were added to the library,a representative “binding curve” is created. FIG. 18A shows, fromexperiment 1 (assessment of affinity measurements using antigentitration), LIBRA-seq scores for one antibody identified from theSARS-CoV-2 convalescent sample using this method. FIG. 18B shows thatthese scores are plotted against the antigen amounts utilized in thescreening library for the titration. FIG. 18C shows comparison of thisexample antibody (shown in black) compared a selection of otherantibodies (colors) identified from this donor. There are a variety ofLIBRA-seq score binding curves that can be used to estimate antigenaffinity. Other measurements can be estimated from these curves, likeEC50 for example.

FIG. 19 shows SARS-CoV-2 S titration with ligand blocking foridentification of potent antibodies. For experiment 3 (assessment ofaffinity measurements combined with ligand blocking), all cellsidentified from the experiment are shown as dots, with LIBRA-seq scorefor ACE2 on the y-axis and LIBRA-seq Score for SARS-CoV-2 S on theX-axis. Each plot shows the LIBRA-seq scores for one of the SARS-CoV-2 Stitration amounts added. These plots are shown from high to low, left toright respectively. With these plots, a SARS-CoV-2 S and ACE2 doublepositive population (shown with an arrow) can be identified, along witha SARS-CoV-2 S positive/ACE2 negative population (shown with an arrow).This population represents cells that have ligand blockingfunctionality. Further, since a titration of Spike was included, cellsthat show high scores for spike added in lower amounts and are alsonegative for ACE2 can be identified (shown in red circle). Thispopulation of cells can be highly potent, ACE2 blocking antibodies.

FIGS. 20A-20C show differentiation of CD4 binding site-directedantibodies using flow cytometry. FIG. 20A shows schematic ofproof-of-concept experiment where VRCO1 (red, left) recognizes theCD4-binding-site of HIV envelope (gray) which blocks its interactionwith CD4 ligand (green). VRC34 (purple, right) recognizes a site on theHIV envelope protein which does not block this interaction. FIGS.20B-20C show binding of soluble HIV envelope and CD4 proteins to VRCO1and VRC34-expressing Ramos B cells lines measured by flow cytometry. TheInfluenza-specific FE53 Ramos B cell line is shown as negative control.

FIG. 21 shows flow sorting strategy to identify virus-specificantibodies from donor 45. To demonstrate the ability of LIBRA-seq toidentify ligand-blocking antibody sequences from an HIV infectionsample, a panel of 3 oligo-labeled HIV envelope antigens andoligo-labeled soluble CD4 antigen were utilized to stain PBMCs from NIHDonor 45. NIH Donor 45 was chosen for investigation due to the number ofpreviously characterized CD4bs antibody lineages identified in thisdonor.

FIG. 22 shows distribution of LIBRA-seq scores identifies cd4bs-specificb cell sequences from donor 45. Plot of maximum LIBRA-seq score (LSS)for a single HIV antigen used in the experiment vs. CD4 LSS where everydot represents a single cell. Cells considered HIV Ag+CD4-(shaded red,defined as having an HIV Ag LSS>1 and a CD4 LSS<1) are predicted beCD4bs-specific. Conversely, Cells considered HIV Ag+CD4+ (shaded purple,defined as having an HIV Ag LSS>1 and a CD4 LSS>1) are predicted torecognize the HIV envelope protein outside the CD4bs.

DETAILED DESCRIPTION

Disclosed herein are high-throughput systems and methods for thedetection of ligand-blocking antibodies and for determining antibodypotency.

Reference will now be made in detail to the embodiments of theinvention, examples of which are illustrated in the drawings and theexamples. This invention may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. The term “comprising” andvariations thereof as used herein is used synonymously with the term“including” and variations thereof and are open, non-limiting terms.Although the terms “comprising” and “including” have been used herein todescribe various embodiments, the terms “consisting essentially of” and“consisting of” can be used in place of “comprising” and “including” toprovide for more specific embodiments and are also disclosed. As used inthis disclosure and in the appended claims, the singular forms “a”,“an”, “the”, include plural referents unless the context clearlydictates otherwise.

The following definitions are provided for the full understanding ofterms used in this specification.

Terminology

The term “about” as used herein when referring to a measurable valuesuch as an amount, a percentage, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

As used herein, the terms “may,” “optionally,” and “may optionally” areused interchangeably and are meant to include cases in which thecondition occurs as well as cases in which the condition does not occur.Thus, for example, the statement that a formulation “may include anexcipient” is meant to include cases in which the formulation includesan excipient as well as cases in which the formulation does not includean excipient.

As used herein, the term “subject” or “host” can refer to livingorganisms such as mammals, including, but not limited to humans,livestock, dogs, cats, and other mammals. Administration of thetherapeutic agents can be carried out at dosages and for periods of timeeffective for treatment of a subject. In some embodiments, the subjectis a human

“Nucleotide,” “nucleoside,” “nucleotide residue,” and “nucleosideresidue,” as used herein, can mean a deoxyribonucleotide orribonucleotide residue, or other similar nucleoside analogue. Anucleotide is a molecule that contains a base moiety, a sugar moiety anda phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate). There are manyvarieties of these types of molecules available in the art and availableherein.

The term “polynucleotide” refers to a single or double stranded polymercomposed of nucleotide monomers.

The method and the system disclosed here including the use of primers,which are capable of interacting with the disclosed nucleic acids, suchas the antigen barcode as disclosed herein. In certain embodiments theprimers are used to support DNA amplification reactions. Typically, theprimers will be capable of being extended in a sequence specific mannerExtension of a primer in a sequence specific manner includes any methodswherein the sequence and/or composition of the nucleic acid molecule towhich the primer is hybridized or otherwise associated directs orinfluences the composition or sequence of the product produced by theextension of the primer. Extension of the primer in a sequence specificmanner therefore includes, but is not limited to, PCR, DNA sequencing,DNA extension, DNA polymerization, RNA transcription, or reversetranscription. Techniques and conditions that amplify the primer in asequence specific manner are preferred. In certain embodiments theprimers are used for the DNA amplification reactions, such as PCR ordirect sequencing. It is understood that in certain embodiments theprimers can also be extended using non-enzymatic techniques, where forexample, the nucleotides or oligonucleotides used to extend the primerare modified such that they will chemically react to extend the primerin a sequence specific manner Typically, the disclosed primers hybridizewith the disclosed nucleic acids or region of the nucleic acids or theyhybridize with the complement of the nucleic acids or complement of aregion of the nucleic acids.

The term “amplification” refers to the production of one or more copiesof a genetic fragment or target sequence, specifically the “amplicon”.As it refers to the product of an amplification reaction, amplicon isused interchangeably with common laboratory terms, such as “PCRproduct.”

The term “polypeptide” refers to a compound made up of a single chain ofD- or L-amino acids or a mixture of D- and L-amino acids joined bypeptide bonds.

As used herein, the term “antigen” refers to a molecule that is capableof stimulating an immune response such as by production of antibodiesspecific for the antigen. Antigens of the present invention can be, forexample, an antigen from human immunodeficiency virus (HIV), an antigenfrom influenza virus, or an antigen from respiratory syncytial virus(RSV). Antigens of the present invention can also be, for example, ahuman antigen (e.g. an oncogene-encoded protein).

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to specifically interact withthe HIV virus, such that the HIV viral infection is prevented,inhibited, reduced, or delayed. The antibodies can be tested for theirdesired activity using the in vitro assays described herein, or byanalogous methods, after which their in vivo therapeutic and/orprophylactic activities are tested according to known clinical testingmethods. There are five major classes of human immunoglobulins: IgA,IgD, IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 andIgA-2. One skilled in the art would recognize the comparable classes formouse. The heavy chain constant domains that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu, respectively.

Each antibody molecule is made up of the protein products of two genes,heavy-chain gene and light-chain gene. The heavy-chain gene isconstructed through somatic recombination of V, D, and J gene segments.In human, there are 51 VH, 27 DH, 6 JH, 9 CH gene segments on humanchromosome 14. The light-chain gene is constructed through somaticrecombination of V and J gene segments. There are 40 Vκ, 31 Vλ, 5 Jκ, 4Jλ. gene segments on human chromosome 14 (80 VJ). The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The “lightchains” of antibodies from any vertebrate species can be assigned to oneof two clearly distinct types, called kappa (κ) and lambda (λ), based onthe amino acid sequences of their constant domains.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity.

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The monoclonal antibodies may also be made by recombinant DNA methods.DNA encoding the disclosed monoclonal antibodies can be readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). Libraries ofantibodies or active antibody fragments can also be generated andscreened using phage display techniques, e.g., as described in U.S. Pat.No. 5,804,440 to Burton et al. and U.S. Pat. No. 6,096,441 to Barbas etal.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross linking antigen.

As used herein, the term “antibody or antigen binding fragment thereof”or “antibody or fragments thereof” encompasses chimeric antibodies andhybrid antibodies, with dual or multiple antigen or epitopespecificities, and fragments, such as F(ab′)2, Fab′, Fab, Fv, sFv, scFvand the like, including hybrid fragments. Thus, fragments of theantibodies that retain the ability to bind their specific antigens areprovided. Such antibodies and fragments can be made by techniques knownin the art and can be screened for specificity and activity according tothe methods set forth in the Examples and in general methods forproducing antibodies and screening antibodies for specificity andactivity (See Harlow and Lane. Antibodies, A Laboratory Manual. ColdSpring Harbor Publications, New York, (1988)).

Also included within the meaning of “antibody or antigen bindingfragment thereof” are conjugates of antibody fragments and antigenbinding proteins (single chain antibodies). Also included within themeaning of “antibody or antigen binding fragment thereof” areimmunoglobulin single variable domains, such as for example a nanobody.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “ligand” refers to a biomolecule or a chemicalentity having a capacity or affinity for binding to a target. A ligandcan include many organic molecules that can be produced by a livingorganism or synthesized, for example, a protein or portion thereof, apeptide, a polysaccharide, an oligosaccharide, a sugar, a glycoprotein,a lipid, a phospholipid, a polynucleotide or portion thereof, anoligonucleotide, an aptamer, a nucleotide, a nucleoside, DNA, RNA, aDNA/RNA chimera, an antibody or fragment thereof, a receptor or afragment thereof, a receptor ligand, a nucleic acid-protein fusion, ahapten, a nucleic acid, a virus or a portion thereof, an enzyme, aco-factor, a cytokine, a chemokine, as well as small molecules (e.g., achemical compound), for example, primary metabolites, secondarymetabolites, and other biological or chemical molecules that are capableof activating, inhibiting, or modulating a biochemical pathway orprocess, and/or any other affinity agent, among others. A ligand cancome from many sources, including libraries, such as small moleculelibraries, phage display libraries, aptamer libraries, or any otherlibrary as would be apparent to one of ordinary skill in the art afterreview of the disclosure of the present disclosure. In some embodiments,the target is an antigen.

In the present invention, “specific for” and “specificity” means acondition where one of the molecules involved in selective binding.Accordingly, an antibody or a ligand that is specific for one antigenselectively binds that antigen and does not substantially recognize orbind other antigens.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific, in another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen, However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

“Pharmaceutically acceptable” component can refer to a component that isnot biologically or otherwise undesirable, i.e., the component may beincorporated into a pharmaceutical formulation of the invention andadministered to a subject as described herein without causingsignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the formulationin which it is contained. When used in reference to administration to ahuman, the term generally implies the component has met the requiredstandards of toxicological and manufacturing testing or that it isincluded on the Inactive Ingredient Guide prepared by the U.S. Food andDrug Administration.

“Pharmaceutically acceptable carrier” (sometimes referred to as a“carrier”) means a carrier or excipient that is useful in preparing apharmaceutical or therapeutic composition that is generally safe andnon-toxic, and includes a carrier that is acceptable for veterinaryand/or human pharmaceutical or therapeutic use. The terms “carrier” or“pharmaceutically acceptable carrier” can include, but are not limitedto, phosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.

The terms “purify” and “purifying” as used herein refers to isolationfrom a biological sample, i.e., blood, plasma, tissues, exosomes,pathogens, or cells. As used herein the term “purified,” when used inthe context of, e.g., an antigen, an antibody, a polypeptide, or anucleic acid, refers to an antigen, an antibody, a polypeptide, or anucleic acid of interest that is at least 60% free, at least 75% free,at least 90% free, at least 95% free, at least 98% free, and even atleast 99% free from other components with which the antigen, theantibody, the polypeptide, the nucleic acid is associated with prior topurification. In some embodiments, an antigen that is not purifiedrefers to said antigen still being associated and/or attached to abiological sample.

As used herein, the terms “treating” or “treatment” of a subjectincludes the administration of a drug to a subject with the purpose ofcuring, healing, alleviating, relieving, altering, remedying,ameliorating, improving, stabilizing or affecting a disease or disorder,or a symptom of a disease or disorder. The terms “treating” and“treatment” can also refer to reduction in severity and/or frequency ofsymptoms, elimination of symptoms and/or underlying cause, andimprovement or remediation of damage.

“Therapeutically effective amount” or “therapeutically effective dose”of a composition refers to an amount that is effective to achieve adesired therapeutic result. Therapeutically effective amounts of a giventherapeutic agent will typically vary with respect to factors such asthe type and severity of the disorder or disease being treated and theage, gender, and weight of the subject. The term can also refer to anamount of a therapeutic agent, or a rate of delivery of a therapeuticagent (e.g., amount over time), effective to facilitate a desiredtherapeutic effect, such as coughing relief. The precise desiredtherapeutic effect will vary according to the condition to be treated,the tolerance of the subject, the agent and/or agent formulation to beadministered (e.g., the potency of the therapeutic agent, theconcentration of agent in the formulation, and the like), and a varietyof other factors that are appreciated by those of ordinary skill in theart. In some instances, a desired biological or medical response isachieved following administration of multiple dosages of the compositionto the subject over a period of days, weeks, or years.

Methods

In some aspects, disclosed herein is a method for simultaneous detectionof an antigen and an antibody that specifically blocks an interactionbetween said antigen and a ligand thereof, comprising:

labeling a plurality of antigens with unique antigen barcodes;

providing a plurality of barcode-labeled antigens to a population ofB-cells to form a mixture;

allowing the plurality of barcode-labeled antigens to bind to thepopulation of B-cells;

labeling one or more ligands to one or more antigens in the plurality ofantigens with unique ligand barcodes;

introducing the one or more ligands to the mixture of the plurality ofbarcode-labeled antigens and the population of B-cells;

washing unbound antigens from the population of B-cells;

separating the B-cells into single cell emulsions;

introducing into each single cell emulsion a unique cell barcode-labeledbead;

preparing a single cell cDNA library from the single cell emulsions;

performing PCR amplification reactions to produce a plurality ofamplicons, wherein the amplicons comprise: 1) the cell barcode and theantigen barcode, 2) the cell barcode and an antibody sequence, and 3)the cell barcode and the ligand barcode, and wherein each ampliconcomprises a unique molecular identifier (UMI);

sequencing the plurality of amplicons;

removing a sequence lacking the cell barcode, the UMI, the ligandbarcode, or the antigen barcode;

aligning the antibody sequence to a reference library of immunoglobulinV, D, J and C sequences;

constructing a first UMI count matrix comprising the cell barcode, theantigen barcode, and the antibody sequence and a second UMI count matrixcomprising the cell barcode, the ligand barcode, and the antibodysequence;

determining a first LIBRA-seq score according to the first UMI countmatrix and a second LIBRA-seq score according to second UMI countmatrix; and

determining that the antibody blocks the interaction between the antigenand the ligand if the first LIBRA-seq score is higher in comparison to afirst reference level and the second LIBRA-seq score is lower incomparison to a second reference level.

In some embodiments, the first reference level is equal to the secondreference level. In some embodiments, the first reference level is about10%, 20%, 30%, 40%, 50%, 60%, 70%, or 90% higher, or at least 2 times, 3times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times,20 times, 40 times, 80 times, 100 times, 500 times higher than thesecond reference level.

Accordingly, in some embodiments, the method of any preceding aspectfurther comprises determining a level of somatic hypermutation of theantibody specifically binding to the antigen.

In some embodiments, the method of any preceding aspect furthercomprises determining a length of a complementarity-determining region(CDR) of the antibody specifically binding to the antigen. The term“complementarity determining region (CDR)” used herein refers to anamino acid sequence of an antibody variable region of a heavy chain orlight chain. CDRs are necessary for antigen binding and determine thespecificity of an antibody. Each variable region typically has threeCDRs identified as CDR1 (CDRH1 or CDRL1, where “H” indicates the heavychain CDR1 and “L” indicates the light chain CDR1), CDR2 (CDRH2 orCDRL2), and CDR3 (CDRH3 or CDRL3). The CDRs may provide contact residuesthat play a major role in the binding of antibodies to antigens orepitopes. Four framework regions, which have more highly conserved aminoacid sequences than the CDRs, separate the CDR regions in the VH or VL.

Accordingly, in some embodiments, the method of any preceding aspectfurther comprises determining a motif of a CDR of the antibodyspecifically binding to the antigen. In some embodiments, the CDR isselected from the group consisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3.

In some embodiments, the method of any preceding aspect furthercomprises identification of IGHV, IGHD, IGHJ, IGKV, IGKJ, IGLV, or IGLJgenes, or combinations thereof, associated with any particularcombination of antigen specificities.

In some embodiments, the method of any preceding aspect furthercomprises identification of mutations in heavy or light FW1, FW2, FW3 orFW4 associated with any particular combination of antigen specificities.

In some embodiments, the method of any preceding aspect furthercomprises identification of overall gene expression profiles or selectup- or down-regulated genes associated with any particular combinationof antigen specificities.

In some embodiments, the method of any preceding aspect furthercomprises identification of surface markers, via, for example,fluorescence-activated cell sorting, or oligo-conjugated antibodiesassociated with any particular combination of antigen specificities

In some embodiments, the method of any preceding aspect furthercomprises identification of any combination of BCR sequence feature (forexample, immunoglobulin gene, sequence motif, or CDR length), geneexpression profile, or surface marker profile associated with anyparticular combination of antigen specificities.

In some embodiments, the method of any preceding aspect furthercomprises training a machine learning algorithm on sequence features,sequence motifs, or encoded sequence properties (such as via Kiderafactors), associated with any particular combination of antigenspecificities for subsequent application to sequenced antibodies lackingantigen specificity information due to not using LIBRA-seq or otherwise.

In some embodiments, the barcode-labeled antigens are labeled with afirst barcode comprising a DNA sequence or an RNA sequence. In someembodiments, the cell barcode-labeled beads are labeled with a secondbarcode comprising a DNA sequence or an RNA sequence.

It should be understood that the barcode described above is conjugatedto the barcode-labeled antigen in a way that are known to one ofordinary skill in the art. Conjugates can be chemically linked to thenucleotide or nucleotide analogs. Such conjugates include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. An oligonucleotide barcode can also beconjugated to an antigen using the Solulink Protein-OligonucleotideConjugation Kit (TriLink cat no. S-9011) according to manufacturer'sinstructions. Briefly, the oligo and protein are desalted, and then theamino-oligo is modified with the 4FB crosslinker, and the biotinylatedantigen protein is modified with S-HyNic. Then, the 4FB-oligo and theHyNic-antigen are mixed together. This causes a stable bond to formbetween the protein and the oligonucleotide. In some embodiments, thecell barcode-labeled beads are labeled with a second barcode comprisinga DNA sequence or an RNA sequence. In some embodiments, the cellbarcode-labeled beads are labeled with a second barcode comprising a DNAsequence. In some embodiments, the cell barcode-labeled beads arelabeled with a second barcode comprising an RNA sequence. In someembodiments, the cell barcode-labeled beads are labeled with a barcodeon the inside of the bead. In some embodiments, the cell barcode-labeledbeads are labeled with a barcode encapsulated within the bead. In someembodiments, the cell barcode-labeled beads are labeled with a barcodeon the outside of the bead.

As used herein, “beads” is not limited to a specific type of bead.Rather, a large number of beads are available and are known to one ofordinary skill in the art. A suitable bead may be selected on the basisof the desired end use and suitability for various protocols. In someembodiments, the bead is or comprises a particle or a bead. In someembodiments, the solid support bead is magnetic. Beads compriseparticles have been described in the prior art in, for example, U.S.Pat. No. 5,084,169, U.S. Pat. No. 5,079,155, U.S. Pat. No. 473,231, andU.S. Pat. No. 8,110,351. The particle or bead size can be optimized forbinding B cell in a single cell emulsion and optimized for thesubsequent PCR reaction.

These oligos, which contain the cell barcode, both: (1) enableamplification of cellular mRNA transcripts through the template switcholigo that is part of the oligo containing the cell barcode, and (2)directly anneal to the antigen barcode-containing oligos from theantigen. In some embodiments, the oligos delivered from the beads havethe general structure:P5_PCR_handle—Cell_barcode—UMI—Template_switch_oligo.

It is noted above that the antibody is determined as specificallybinding an antigen if the LIBRA-seq score of the antibody for theantigen is increased in comparison to a control sample. It should beunderstood herein that between the minimum (y-axis, top) and maximum(y-axis, bottom) LIBRA-seq score for each antigen, the ability of eachof 100 cutoffs was tested for its ability to classify each antibody asantigen positive or negative, where antigen positive is defined ashaving a LIBRA-seq score greater than or equal to the cutoff beingevaluated and antigen negative is defined as having a LIBRA-seq scorebelow the cutoff.

In some embodiments, the antibody sequence comprises an immunoglobulinheavy chain (VDJ) sequence, or an immunoglobulin light chain (VJ)sequence. In some embodiments, the antibody sequence comprises animmunoglobulin heavy chain (VDJ) sequence. In some embodiments, theantibody sequence comprises an immunoglobulin light chain (VJ) sequence.

In some embodiments, the barcode-labeled antigens comprise an antigenfrom a pathogen or an animal. In some embodiments, the barcode-labeledantigens comprise an antigen from a pathogen. In some embodiments, thebarcode-labeled antigens comprise an antigen from an animal. In someembodiments, the animal is a mammal, including, but not limited to,primates (e.g., humans and nonhuman primates), cows, sheep, goats,horses, dogs, cats, rabbits, rats, mice and the like. In someembodiments, the subject is a human

In some embodiments, the antigens are purified before labeling withbarcodes. In some embodiments, the antigens are not purified beforelabeling with barcodes.

Therefore, in some embodiments, the LIBRA-seq disclosed herein can beused for soluble antigens that are not purified. Antigens can bearranged in a plate format for microexpression. Each antigen isexpressed in microculture in a single well on a plate. In someembodiments, a unique barcode is added to each well, barcoded antigensare mixed together, mixed with B cells, and LIBRA-seq is performed asdescribed herein.

In some embodiments, the LIBRA-seq disclosed herein can be used forantigens that are not in soluble form. In some embodiments, a wholevirus is tagged with one or more barcodes. In some embodiments, a wholevirus can use scRNA-seq to exploit the intrinsic diversity withinvariants of the same virus (e.g., different strains of HIV-1). In someembodiments, a pseudovirus can contain internal barcode for LIBRA-seq.In some embodiments, comprehensive mutant virus libraries can begenerated using the methods of (e.g. Dingens et al (2019). An AntigenicAtlas of HIV-1 Escape from Broadly Neutralizing Antibodies DistinguishesFunctional and Structural Epitopes. Immunity, 50(2):520-532.e3). In someembodiments, a whole cell can be tagged by lentiviral transfection withbarcode or CRISPR-based tagging. In some embodiments, amembrane-anchored antigen (in particular, those for membrane proteins),e.g., the one on liposomes is tagged with one or more barcodes.

Antigens can also be formatted into antigen microarrays (for example,VirScan technology, as described in Xu G J, et al. Comprehensiveserological profiling of human populations using a synthetic humanvirome. Science. 2015; 348(6239):aaa0698). DNA microarrays can be usedfollowed by phage display of antigens. In some embodiments herein, aunique barcode is added to each antigen in the microarray.

Antigens of the present invention can also be an antigen from a pathogenor an animal. In some embodiments, the antigen is from a human In someembodiments, the antigen from a human is an antigen encoded by anoncogene. In some embodiments, the antigen encoded by an oncogene canbe, for example, NY-ESO-1, MAGEA-A3, hTERT, Tyrosinase, gp100, MART-1,melanA, beta-catenin, CDC27, hsp70, HLA-A2-R170J, CEA, AFP, PSA,EBV-EBNA, HPV16-E7, MUC-1, HER-2/neu, or Mammaglobin-A.

In some embodiments, the antigen from a pathogen comprises an antigenfrom a virus. In some embodiments, the antigen from a virus comprises anantigen from human immunodeficiency virus (HIV), an antigen frominfluenza virus, or an antigen from respiratory syncytial virus (RSV).

In some embodiments, the antigen from a virus comprises an antigen fromhuman immunodeficiency virus (HIV). In some embodiments, the antigenfrom a virus comprises an antigen from influenza virus. In someembodiments, the antigen from a virus comprises an antigen fromrespiratory syncytial virus (RSV).

In some embodiments, the antigen from HIV comprises an antigen fromHIV-1. In some embodiments, the antigen from HIV comprises an antigenfrom HIV-2. In some embodiments, the antigen from HIV comprises HIV-1Env. In some embodiments, the antigen from influenza virus compriseshemagglutinin (HA). In some embodiments, the antigen from RSV comprisesan RSV F protein. In some embodiments, the antigen is selected from theantigens listed in Table 1.

TABLE 1 Antigen screening library for human B-cell sample analysis. Fora set of pathogens, shown are selected protein targets, number ofstrains, and resulting total number of antigens in the screeninglibrary. Protein # Antigens Pathogen targets # Strains in library CMV gB2 2 Dengue E, prM 5 10 Hepatitis B HBsAg 2 2 Hepatitis C E2, E1E2 2 4HIV-1 gp140, gp120, MPER 3 9 HPV L1 3 3 HSV-1 gB 1 1 Influenza HA, NA *12 Malaria PfCSP 1 1 Measles H, F 1 2 Mumps HN, NP 1 2 Norovirus P 10 10 Rhinovirus VP1 5 5 Rotavirus VP7, VP4 {circumflex over ( )} 8 RSV F,G 4 8 Rubella E1 1 1 Staphylococcus HtsA, SirA, IsdB, SstD 1 4 aureusUPEC Hma, IutA, FyuA, IreA 1 4 Zika E, prM 1 2 *influenza: A (6 HA, 4NA) and B (2 HA); {circumflex over ( )}rotavirus: 6 G, 2 P variants)

In some embodiments, the population of B-cells comprise a memory B-cell,a plasma cell, naïve B cell, an activated B-cell, or a B-cell line. Insome embodiments, the population of B-cells comprise a memory B-cell, aplasma cell, a naïve B cell, an activated B-cell, or a B-cell line. Insome embodiments, the population of B-cells comprise a plasma cell. Insome embodiments, the population of B-cells comprise a naïve B cell. Insome embodiments, the population of B-cells comprise an activatedB-cell. In some embodiments, the population of B-cells comprise a B-cellline.

In some aspects, disclosed herein is a method for simultaneouslyscreening an antigen and an antibody that specifically binds saidantigen, comprising:

-   -   generating a plurality of antigens using an antigen display        technology, wherein each of the plurality of antigens is linked        to a nucleic acid sequence that identifies a particular antigen;    -   providing the plurality of antigens to a population of B-cells;    -   allowing the plurality of antigens to bind to the population of        B-cells;    -   washing unbound antigens from the population of B-cells;    -   separating the B-cells into single cell emulsions;    -   introducing into each single cell emulsion a unique cell        barcode-labeled bead;    -   preparing a single cell cDNA library from the single cell        emulsions;    -   performing PCR amplification reactions to produce a plurality of        amplicons,

wherein the amplicons comprise: 1) the cell barcode and the nucleic acidsequence that identifies the particular antigen, and 2) the cell barcodeand an antibody sequence, and

wherein each amplicon comprises a unique molecular identifier (UMI);

-   -   sequencing the plurality of amplicons;    -   removing a sequence lacking the cell barcode, the UMI, or the        nucleic acid sequence that identifies the particular antigen;    -   aligning the antibody sequence to a reference library of        immunoglobulin V, D, J and C sequences;    -   constructing a UMI count matrix comprising the cell barcode, the        nucleic acid sequence that identifies the particular antigen,        and the antibody sequence;    -   determining a LIBRA-seq score;    -   determining the nucleic acid sequence that identifies the        particular antigen; and    -   determining that the antibody specifically binds an antigen if        the LIBRA-seq score of the antibody for the antigen is higher        than a reference level.

In some embodiments, the amplicon comprising the cell barcode andnucleic acid sequence that identifies a particular antigen and theamplicon comprising the cell barcode and an antibody sequence areseparate amplicons. In some embodiments, the antibody sequence can be aheavy chain sequence and/or a light chain sequence. In some embodiments,the antibody sequence is a heavy chain sequence. In some embodiments,the antibody sequence is a light chain sequence.

In some embodiments, the antigen display technology comprises a ribosomedisplay technology.

In some embodiments, the nucleic acid sequence that identifies aparticular antigen is a coding nucleic acid. In some embodiments, thenucleic acid sequence that identifies a particular antigen is a codingnucleic acid sequence that is covalently linked to the antigen. In someembodiments, the nucleic acid sequence that identifies a particularantigen is a nucleic acid sequence from the DNA (for example, viralDNA). In some embodiments, the nucleic acid sequence that identifies aparticular antigen is an exon sequence. In some embodiments, the nucleicacid sequence that identifies a particular antigen is a transcriptsequence. In some embodiments, the nucleic acid sequence that identifiesa particular antigen is an antigen barcode.

In some embodiments, the antigen display technology comprises a phagedisplay technology, a yeast display technology, or a ribosome displaytechnology. In some embodiments, the term “phage display technology,” asused herein is meant to refer to those forms described more fully in H.Benjamin Larman, Nat Biotechnol. 2011 June; 29(6): 535-541., and U.S.Pat. No. 6,017,732, incorporated herein by reference for all purposes.In some embodiments, the term “ribosome display technology,” as usedherein is meant to refer to those forms described more fully in Zhu etal., Nat Biotechnol. 2013 April; 31(4): 331-334, WO200¹/₇5097 and U.S.Pat. No. 774,557, incorporated herein by reference for all purposes. Seealso, Science. 2015 June 5; 348(6239).

In some embodiments, B cells are purified from a sample of interest (forexample, tumor infiltrates); non B cell populations from the sample ofinterest are obtained (for example, consisting of many tumor cells) tosequence exome; an antigen screening library is made using ribosomedisplay of exons; and B cells are screened against the antigen screeninglibrary.

In some aspects, disclosed herein is a method for determining a bindingpotency of an antibody to an antigen, comprising:

labeling a plurality of antigens with unique antigen barcodes;

providing a plurality of barcode-labeled antigens to a population ofB-cells;

allowing the plurality of barcode-labeled antigens to bind to thepopulation of B-cells;

washing unbound antigens from the population of B-cells;

separating the B-cells into single cell emulsions;

introducing into each single cell emulsion a unique cell barcode-labeledbead;

preparing a single cell cDNA library from the single cell emulsions;

performing PCR amplification reactions to produce a plurality ofamplicons, wherein the amplicons comprise: 1) the cell barcode and theantigen barcode, and 2) the cell barcode and an antibody sequence, andwherein each amplicon comprises a unique molecular identifier (UMI);

sequencing the plurality of amplicons;

removing a sequence lacking the cell barcode, the UMI, or the antigenbarcode;

aligning the antibody sequence to a reference library of immunoglobulinV, D, J and C sequences;

constructing a UMI count matrix comprising the cell barcode, the antigenbarcode, and the antibody sequence;

determining a LIBRA-seq score; and

determining that the antibody has a high binding potency to the antigenif the LIBRA-seq score of the antibody for the antigen is higher than areference level.

The term “binding potency” used herein refers to the concentration oramount of an antibody required to produce a certain effect, such asbinding to a certain amount of antigen. The antibody-antigen bindingpotency can be measured using a variety of techniques, for exampleELISA, SPR, BLI, etc.

In some embodiments, the methods herein are used for epitope mapping. Insome embodiments, the method of any preceding aspect further comprises astep for pre-filtering for antigen-bound B cells.

EXAMPLES

The following examples are set forth below to illustrate the systems,methods, and results according to the disclosed subject matter. Theseexamples are not intended to be inclusive of all aspects of the subjectmatter disclosed herein, but rather to illustrate representative methodsand results. These examples are not intended to exclude equivalents andvariations of the present invention which are apparent to one skilled inthe art.

Example 1. LIBRA-seq for Antibody Discovery with Ligand Blocking

In some antibody discovery efforts, it is important to identifyfunctional (as opposed to binding-only) antibodies, and in many casesthe function of interest is the identification of antibodies that canblock antigen interactions with its cognate ligand. Rather thanperforming standard screens for antigen-binding antibodies, followed byligand-blocking assays for select antibodies, disclosed herein is amethod adapting LIBRA-seq to simultaneously obtain information on theligand-blocking abilities of identified B cells, from the samesequencing experiment. To achieve this, for a given antigen, the knowncognate ligand can also be labeled with a unique barcode. B cells arefirst mixed with the antigens from the screening library, followed bymixing with any ligand(s) (FIG. 5 ). Upon sequencing, B cells that havea high LIBRA-seq score for the given antigen but a low LIBRA-seq scorefor the corresponding ligand indicates antigen-ligand binding isdecreased when the antigen is bound to the BCRs for the given B cell(FIG. 6 ).

LIBRA-seq experiments are performed with barcoded ligands and theirrespective barcoded antigen partners in the screening library, alongwith B cell lines both for antibodies that do and do not block ligandbinding for the same antigen. For example, soluble CD4 is used as aligand for the HIV-1 Env antigen, along with VRCO1 B cells (which canblock Env-CD4 binding) and another HIV-1 B cell line (e.g., 2G12, whichcannot not block Env-CD4 binding). In these experiments, severalparameters are varied and evaluated, including ligand amounts, number ofoligos per ligand molecule, barcode variation, ligand-to-antigen ratio,etc. Ultimately, these experiments inform the selection of parametersthat optimize the LIBRA-seq ability to accurately identifyligand-blocking antibodies using a benchmarking system of knownantigen-ligand and B cell line combinations.

Example 2. LIBRA-seq for High-throughput Antigen Screening and de novoAntigen Discovery

The typical LIBRA-seq platforms require oligo-tagging of a set of targetantigens. LIBRA-seq can be extended to use antigen display technologies(such as phage, yeast, or ribosome display), such that for each given Bcell, all bound displayed antigens can be sequenced simultaneously. Forexample, ribosome display for protein antigens is an establishedtechnique (e.g., Zhu et al., Nat Biotechnol. 2013 April; 31(4):331-334.). LIBRA-seq can utilize display technologies by combining withthe B cell side of the analysis, resulting in paired BCRsequence-antigen identity information (FIG. 7 ). In particular, thisapproach allows sequencing of genetic material for the displayedantigens. For example, with phage display, the phage DNA can besequenced; for ribosome display, the antigen mRNA is tethered to theantigen protein that it encodes through lack of a stop codon. Theadvantage of the display technologies is that an immensely larger set ofantigens can be screened in a single experiment (tens of thousands topotentially billions), compared to the dozens to hundreds of antigensthat are screened with the traditional approach that requires productionand purification for each individual antigen. The antigen displaylibrary can be targeted (e.g., multiple variants of the same antigenprotein) or it can be general (e.g., thousands of human proteins).Importantly, this approach enables antigen discovery: B cells can bescreened against large-scale human proteome, virome, microbiome etc.antigen libraries without a pre-conceived notion about the specificantigen target that these B cells can target, thus enabling thesimultaneous discovery of both antibodies and antigens.

Example 3. LIBRA-seq for Antibody-antigen Potency Determination

One of the primary goals in many antibody discovery efforts is toefficiently select for high-potency as opposed to low-potency antibodiesthat are specific to a target antigen. It is therefore of value toenable LIBRA-seq to estimate, or at least rank-order, B cell potency fora target antigen. To achieve this, several approaches are disclosedherein.

Qualitative potency estimates. For many target antigens of biomedicalinterest, there already exist antibodies for which potency measurementscan be performed using standard techniques, such as biolayerinterferometry. To investigate the ability of LIBRA-seq to discriminateB cells based on the potency of the BCRs for a target antigen, LIBRA-seqis evaluated to score B cells that have different affinities for thesame antigen. In particular, the LIBRA-seq scores are compared for thedifferent influenza HA antigens from the screening library against thedifferent influenza antibodies represented in the B cell lines, sinceall of these antibody-antigen pairs have known potency. In addition, Bcells expressing the BCRs for a low-potency germline-reverted version ofFe53 are used. These experiments are performed at different B cellratios, to interrogate the limits of LIBRA-seq detection ofantigen-specific B cells as a function of both potency and relativeabundance. Upon sequencing, the LIBRA-seq scores for a given antigen arehigher for higher-potency B cells and lower for lower-potency B cells.Ultimately, for antigens for which a known antibody exists, theLIBRA-seq experiments can be set up to use a B cell control with knownantigen potency, in order to prioritize newly identified B cells basedon their LIBRA-seq scores compared to the control. The data in FIG. 8show lower scores for VRCO1 cells against the lower-potency D368Rmutation compared to wildtype antigen, indicating that LIBRA-seq scorescan be used as a relative indicator of antibody-antigen potency.

Quantitative potency estimates. The ability of LIBRA-seq for estimatingantibody-antigen potency in a more quantitative manner is also explored.To achieve this, a given antigen is aliquoted and barcoded withdifferent unique barcodes (one barcode per aliquot). A titration seriesare then performed, such that the B cells are be mixed with different(but pre-defined) amounts from each of the barcoded aliquots for thegiven antigen. Upon sequencing, a pseudo-potency measurement can beobtained for a given B cell by fitting a curve to the LIBRA-seq scoresfor the different barcoded aliquots for the same antigen (FIG. 9 ). Thisexperimental setup is used with the different antigens from thescreening library discussed above, followed by a comparison of theresulting pseudo-potency measurements for the respectiveantigen-specific B cell lines to the measured potencies for therespective antibodies. The effect of a number of parameters areevaluated (including the number of unique barcodes per antigen (e.g.,between 4 and 10), amounts for each barcoded variant of an antigen,etc.) on LIBRA-seq potency estimation accuracy. This approach allows forcomparisons between different antibodies and antigens, and even betweenmultiple LIBRA-seq experiments, without the need for a knownantibody-antigen potency control, in order to enable the prioritizationof B cells based on the potency estimates for any given target antigen.

Example 4. LIBRA-seq with Pre-filtering for Antigen-bound B Cells

While the LIBRA-seq antigen screening library can be adapted to eachspecific sequencing experiment, within any given sample, there can be asubstantial number of B cells specific to antigens that are not includedin the screening library (antigen-specific B cells for a given antigenare typically low frequency). When such B cells are included in thesequencing experiment, only their BCR sequence information is obtained,without any information on their antigen specificity, since there are nomatching antigens in the antigen screening library for these B cells.

To address this, strategies are explored for focusing the sequencingspecifically toward antigen-bound B cells (for which both antibodysequence and antigen specificity information can be obtained). Namely,antigen-positive cells are sorted, using the following strategies forfluorescently labeling the antigens in the antigen screening library:(a) fluorescently labeled streptavidin and (b) fluorescently labeledoligo barcodes, synthesized by Sigma Aldrich with four internalfluorescein-dTs and a 5′ amino C6 linker.

With each of the two strategies, the fluorescently labeled antigens areassociated with a single color (independent of antigen). Before sampleprocessing (for example , 10×) and NGS, the B cell-antigen mixtures aresubjected to fluorescence-activated cell sorting (FACS), to select for Bcells that are bound to the barcoded antigens. For sorting, cells arecounted and viability are assessed using Trypan Blue, washed with DPBSsupplemented with 1% Bovine serum albumin (DPBS-BSA) throughcentrifugation at 300 g for 7 minutes, and resuspended in DPBS-BSA andstained with a variety of cell markers including CD3-APCCy7, IgG-FITC,CD19-BV711, CD14-V500, and LiveDead-V500. The fluorescently labeledantigen-oligo conjugates are added to the stain, so antigen-specificsorting can occur. After staining in the dark for 30 minutes at roomtemperature, cells are washed 3 times with DPBS-BSA at 300 g for 7minutes. Then, cells are resuspended in DPBS-BSA and analyzed and/orsorted on the flow cytometer/cell sorter. Antigen-specific B cells aretaken as: live, CD14− CD3− CD19+ IgG+, and positive for the PE color,with which all barcoded antigens are labeled (FIGS. 11A, 11B, and 12 ).

This method enables the determination of antigen-bound B cells for anyantigen in the screening library. For increased robustness, eachbarcoded antigen can be labeled with two different colors, so that onlyB cells that are double-positive for both colors are retained. Thefluorescent labeling of the antigens does not affect the ability todistinguish between different antigen barcodes in the subsequentsingle-cell analysis and NGS sequencing experiments. In essence, thefluorescent labeling helps select antigen-bound B cells, whereas thesubsequent sequencing of the antigen oligo barcodes helps determinewhich antigens are bound to each of the sorted antigen-bound B cells.The primary difference from non-fluorescent antigen experiments is thatonly antigen-bound B cells are sequenced here, thus increasing theefficiency and the amount of information that can be obtained from asingle sequencing experiment. This filtering step focuses in on rarepopulations of antigen-specific B cells, ensuring that maximuminformation is extracted for B cells that have specificity for any ofthe antigens in the screening library (FIG. 10 ). If desired,non-antigen-specific B cells from the same sample can also be subjectedto separate prep (for example 10×) and sequencing, to maximize theamount of BCR sequence information extracted from a given sample.

Example 5. LIBRA-Seq Methods

Following a LIBRA-seq experiment, there are 2 resulting pairs of FASTQfiles: (1) B cell receptor libraries (containing heavy and light chaincontigs), and (2) antigen barcode libraries (containingantigen-identifying DNA barcode sequences from the antigen screeninglibrary). In some embodiments, it should be understood that the methodsdescribed herein are for uniting the information from these twosequencing libraries. Accordingly, in some embodiments, the above notedstep of removing a sequence lacking the cell barcode, the UMI, or theantigen barcode is for removing a sequence from the antigen barcodelibrary lacking the cell barcode, the UMI, or the antigen barcode. Themethods describe here are for processing the antigen barcodes. Theprocessing serves two purposes: (1) quality control and annotation ofsequenced reads, and (2) identification of binding signal from theannotated sequenced reads. Before the following steps are carried out,the BCR libraries are processed in order to determine the list of cellbarcodes that have a VDJ sequence.

Processing of antigen barcode reads and BCR sequence contigs. A pipelineshown herein takes paired-end fastq files of oligo libraries as input,processes and annotates reads for cell barcode, UMI, and antigenbarcode, and generates a cell barcode - antigen barcode UMI countmatrix. BCR contigs can be processed using cellranger (10X Genomics)using GRCh38 as reference. For the antigen barcode libraries, initialquality and length filtering is carried out by fastp (Chen et al., 2018)using default parameters for filtering. This results in onlyhigh-quality reads being retained in the antigen barcode library. In ahistogram of insert lengths, this results in a sharp peak of theexpected insert size of 52-54. Fastx_collapser is then used to groupidentical sequences and convert the output to deduplicated fasta files.Then, having removed low-quality reads, just the R2 sequences wereprocessed, as the entire insert is present in both R1 and R2. Eachunique R2 sequence (or R1, or the consensus of R1 and R2) was processedone by one using the following steps:

(1) The reverse complement of the R2 sequence is determined (Skip step 1if using R1).

(2) The sequence is screened for possessing an exact (or near exact)match to any of the valid cell barcodes present in thefiltered_contig.fasta file output by cell ranger during processing ofBCR V(D)J fastq files. Sequences without a BCR-associated cell barcodeare discarded.

(3) The 10 bases immediate 3′ to the cell barcode are annotated as theread's UMI.

(4) The remainder of the sequence 3′ to the UMI is screened for a 13 or15 bp sequence with a hamming distance of 0, 1, or 2 to any of theantigen barcodes used in the screening library. Following thisprocessing, only sequences around the expected lengths are retained (thelengths of sequences can be from more than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 bases shorter to more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 bases longer than the expected lengths), thus allowing for adeletion, an insertion outside the cell barcode, or bases flanking thecell barcode.

This general process requires that sequences possess all elements neededfor analysis (cell barcode, UMI, and antigen barcode), but is permissiveto insertions or deletions in the TSO region between the UMI and antigenbarcode. After processing each sequence one-by-one, cell barcode - UMI -antigen barcode collisions are screened. Any cell barcode - UMIcombination (indicative of a unique oligo molecule) that has multipleantigen barcodes associated with it is removed. A cell barcode - antigenbarcode UMI count matrix is then constructed, which served as the basisof subsequent analysis. Additionally, the BCR contigs are aligned(filtered_contigs.fasta file output by Cellranger) to IMGT referencegenes using HighV-Quest (Alamyar et al., 2012). The output ofHighV-Quest is parsed using ChangeO (Gupta et al., 2015), and mergedwith the UMI count matrix.

The above stated procedure can be summarized as the following steps:

1) Remove low quality reads;

2) Remove reads too long or too short to be a valid antigen barcode readcontaining a cell barcode, UMI, and antigen barcode;

3) For each quality read, annotate:

-   -   a. Cell barcode,    -   b. UMI    -   c. Antigen barcode, allowing for sequencing/PCR errors by using        a hamming distance threshold.

Determination of LIBRA-seq Score. Starting with the UMI count matrix,all counts of more than one UMIs (for example, more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. UMIs) canbe set to 0, with the idea that these low counts can be attributed tonoise. After this, the UMI count matrix was subset to contain only cellswith a count of one or more UMIs than the minimum value in the abovenoted step of noise filtering for at least 1 antigen. The centered-logratios (CLR) of each antigen UMI count for each cell were thencalculated (Mimitou et al., 2019; Stoeckius et al., 2017, 2018). BecauseUMI counts were on different scales for each antigen, due todifferential oligo loading during oligo-antigen conjugation, the CLRsUMI counts were rescaled using the StandardScaler method in scikit learn(Pedregosa and Varoquaux, 2011). Lastly, A correction procedure wasperformed to the z-score-normalized CLRs from UMI counts of 0, settingthem to the minimum for each antigen for donor NIAID 45 and N90experiments, and to -1 for the Ramos B cell line experiment. TheseCLR-transformed, Z-score-normalized, corrected values served as thefinal LIBRA-seq scores. LIBRA-seq scores were visualized using Cytobank(Kotecha et al., 2010).

Identification of sequence feature—antigen specificity associations.Following determination of LIBRA-seq scores (above), and becauseantibody sequence is united with antigen specificity (in the form of aLIBRA-seq score), sequence-specificity associations can be made.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A method for simultaneous detection of an antigenand an antibody that specifically blocks an interaction between saidantigen and a ligand thereof, comprising: labeling a plurality ofantigens with unique antigen barcodes; providing a plurality ofbarcode-labeled antigens to a population of B-cells to form a mixture;allowing the plurality of barcode-labeled antigens to bind to thepopulation of B-cells; labeling one or more ligands to one or moreantigens in the plurality of antigens with unique ligand barcodes;introducing the one or more ligands to the mixture of the plurality ofbarcode-labeled antigens and the population of B-cells; washing unboundantigens from the population of B-cells; separating the B-cells intosingle cell emulsions; introducing into each single cell emulsion aunique cell barcode-labeled bead; preparing a single cell cDNA libraryfrom the single cell emulsions; performing PCR amplification reactionsto produce a plurality of amplicons, wherein the amplicons comprise: 1)the cell barcode and the antigen barcode, 2) the cell barcode and anantibody sequence, and 3) the cell barcode and the ligand barcode, andwherein each amplicon comprises a unique molecular identifier (UMI);sequencing the plurality of amplicons; removing a sequence lacking thecell barcode, the UMI, the ligand barcode, or the antigen barcode;aligning the antibody sequence to a reference library of immunoglobulinV, D, J and C sequences; constructing a first UMI count matrixcomprising the cell barcode, the antigen barcode, and the antibodysequence and a second UMI count matrix comprising the cell barcode, theligand barcode, and the antibody sequence; determining a first LIBRA-seqscore according to the first UMI count matrix and a second LIBRA-seqscore according to second UMI count matrix; and determining that theantibody blocks the interaction between the antigen and the ligand ifthe first LIBRA-seq score is higher in comparison to a first referencelevel and the second LIBRA-seq score is lower in comparison to a secondreference level.
 2. The method of claim 1, wherein the barcode-labeledantigens are labeled with a first barcode comprising a DNA sequence oran RNA sequence.
 3. The method of claim 1 or claim 2, wherein the cellbarcode-labeled beads are labeled with a second barcode comprising a DNAsequence or an RNA sequence.
 4. The method of any one of claims 1 to 3,wherein the antibody sequence comprises an immunoglobulin heavy chain(VDJ) sequence, or an immunoglobulin light chain (VJ) sequence.
 5. Themethod of any one of claims 1 to 4, wherein the barcode-labeled antigenscomprise a membrane-anchored antigen.
 6. The method of any one of claims1 to 5, wherein the barcode-labeled antigens comprise an antigen from apathogen or an animal.
 7. The method of claim 6, wherein the antigen isnot purified.
 8. The method of claim 6 or claim 7, wherein the antigenfrom a pathogen comprises an antigen from a virus.
 9. The method ofclaim 8, wherein the antigen from a virus comprises an antigen fromhuman immunodeficiency virus (HIV), an antigen from influenza virus, oran antigen from respiratory syncytial virus (RSV).
 10. The method of anyone of claims 1 to 9, further comprising determining a level of somatichypermutation of the antibody specifically binding to the antigen. 11.The method of any one of claims 1 to 10, further comprising determininga length of a complementarity-determining region (CDR) of the antibodyspecifically binding to the antigen.
 12. The method of any one of claims1 to 11, further comprising determining a motif of a CDR of the antibodyspecifically binding to the antigen.
 13. The method of claim 11 or 12,wherein the CDR is selected from the group consisting of CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3.
 14. A method for simultaneouslyscreening an antigen and an antibody that specifically binds saidantigen, comprising: generating a plurality of antigens using an antigendisplay technology, wherein each of the plurality of antigens is linkedto a nucleic acid sequence that identifies a particular antigen;providing the plurality of antigens to a population of B-cells; allowingthe plurality of antigens to bind to the population of B-cells; washingunbound antigens from the population of B-cells; separating the B-cellsinto single cell emulsions; introducing into each single cell emulsion aunique cell barcode-labeled bead; preparing a single cell cDNA libraryfrom the single cell emulsions; performing PCR amplification reactionsto produce a plurality of amplicons, wherein the amplicons comprise: 1)the cell barcode and the nucleic acid sequence that identifies theparticular antigen, and 2) the cell barcode and an antibody sequence,and wherein each amplicon comprises a unique molecular identifier (UMI);sequencing the plurality of amplicons; removing a sequence lacking thecell barcode, the UMI, or the nucleic acid sequence that identifies theparticular antigen; aligning the antibody sequence to a referencelibrary of immunoglobulin V, D, J and C sequences; constructing a UMIcount matrix comprising the cell barcode, the nucleic acid sequence thatidentifies the particular antigen, and the antibody sequence;determining a LIBRA-seq score; determining the nucleic acid sequencethat identifies the particular antigen; and determining that theantibody specifically binds an antigen if the LIBRA-seq score of theantibody for the antigen is higher than a reference level.
 15. Themethod of claim 14, wherein the plurality of antigens are labeled with afirst barcode comprising a DNA sequence or an RNA sequence.
 16. Themethod of claim 14 or claim 15, wherein the cell barcode-labeled beadsare labeled with a second barcode comprising a DNA sequence or an RNAsequence.
 17. The method of any one of claims 14 to 16, wherein theantibody sequence comprises an immunoglobulin heavy chain (VDJ)sequence, or an immunoglobulin light chain (VJ) sequence.
 18. The methodof any one of claims 14 to 17, wherein the plurality of antigenscomprise an antigen from a pathogen or an animal.
 19. The method ofclaim 18, wherein the antigen is not purified.
 20. The method of claim18 or claim 19, wherein the antigen from a pathogen comprises an antigenfrom a virus.
 21. The method of claim 20, wherein the antigen from avirus comprises an antigen from human immunodeficiency virus (HIV), anantigen from influenza virus, or an antigen from respiratory syncytialvirus (RSV).
 22. The method of any one of claims 14 to 21, furthercomprising determining a level of somatic hypermutation of the antibodyspecifically binding to the antigen.
 23. The method of any one of claims14 to 22, further comprising determining a length of acomplementarity-determining region (CDR) of the antibody specificallybinding to the antigen.
 24. The method of any one of claims 14 to 23,further comprising determining a motif of a CDR of the antibodyspecifically binding to the antigen.
 25. The method of claim 23 or 24,wherein the CDR is selected from the group consisting of CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3.
 26. The method of any one of claims 14to 25, wherein the antigen display technology comprises a ribosomedisplay technology.
 27. A method for determining a binding potency of anantibody to an antigen, comprising: labeling a plurality of antigenswith unique antigen barcodes; providing a plurality of barcode-labeledantigens to a population of B-cells; allowing the plurality ofbarcode-labeled antigens to bind to the population of B-cells; washingunbound antigens from the population of B-cells; separating the B-cellsinto single cell emulsions; introducing into each single cell emulsion aunique cell barcode-labeled bead; preparing a single cell cDNA libraryfrom the single cell emulsions; performing PCR amplification reactionsto produce a plurality of amplicons, wherein the amplicons comprise: 1)the cell barcode and the antigen barcode, and 2) the cell barcode and anantibody sequence, and wherein each amplicon comprises a uniquemolecular identifier (UMI); sequencing the plurality of amplicons;removing a sequence lacking the cell barcode, the UMI, or the antigenbarcode; aligning the antibody sequence to a reference library ofimmunoglobulin V, D, J and C sequences; constructing a UMI count matrixcomprising the cell barcode, the antigen barcode, and the antibodysequence; determining a LIBRA-seq score; and determining that theantibody has a high binding potency to the antigen if the LIBRA-seqscore of the antibody for the antigen is higher than a reference level.28. The method of claim 27, wherein the barcode-labeled antigens arelabeled with a first barcode comprising a DNA sequence or an RNAsequence.
 29. The method of claim 27 or claim 28, wherein the cellbarcode-labeled beads are labeled with a second barcode comprising a DNAsequence or an RNA sequence.
 30. The method of any one of claims 27 to29, wherein the antibody sequence comprises an immunoglobulin heavychain (VDJ) sequence, or an immunoglobulin light chain (VJ) sequence.31. The method of any one of claims 27 to 30, wherein thebarcode-labeled antigens comprise an antigen from a pathogen or ananimal.
 32. The method of claim 31, wherein the antigen is not purified.33. The method of claim 31 or claim 32, wherein the antigen from apathogen comprises an antigen from a virus.
 34. The method of claim 33,wherein the antigen from a virus comprises an antigen from humanimmunodeficiency virus (HIV), an antigen from influenza virus, or anantigen from respiratory syncytial virus (RSV).
 35. The method of anyone of claims 27 to 34, further comprising determining a level ofsomatic hypermutation of the antibody specifically binding to theantigen.
 36. The method of any one of claims 27 to 35, furthercomprising determining a length of a complementarity-determining region(CDR) of the antibody specifically binding to the antigen.
 37. Themethod of any one of claims 27 to 36, further comprising determining amotif of a CDR of the antibody specifically binding to the antigen. 38.The method of claim 36 or 37, wherein the CDR is selected from the groupconsisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.